The present invention relates to a method for separating and purifying nucleic acids to be used for the genome analysis or the gene manipulation. In more detail, it relates to a method for separating long chain nucleic acids represented by particular DNA contained in the cells of animals, human, etc. that are effective for the gene therapy to be utilized for the therapy of genetic disease etc. due to DNA abnormality, by utilizing liquid chromatography, for example, plasmids, in the bacteria, organelle DNA, phage DNA, etc.
In recent years, the gene therapy is attracting an attention, and a method for separating and purifying a large quantity of long chain nucleic acids such as plasmids and DNA fragments to be used for gena therapy in simpler way and in shorter time is desired eagerly.
Here, for using nucleic acids for the therapy of human, it is desirable that the nucleic acids can be separated and purified keeping the same structure (higher-order structure) as that when they exist in organisms. Here, since the enzyme reaction is utilized for the recombination of nucleic acids, the nucleic acids are required to have been separated and purified to the extent that they can become the substrate for reaction. Also, in order to avoid the adverse effect of impurities on human body, the nucleic acids are required to have been separated and purified up to high purity.
For separating and purifying long chain nucleic acids such as DNAs and plasmids contained in the cells and bacteria, chemical treatment methods have been used most frequently, so far.
Among various long chain nucleic acids used for the gene therapy, in particular, plasmid is currently utilized in many cases, because of limited cleavage sites by particular restriction enzyme and relatively easy recombination manipulation. In the following, a general example of purifying a plasmid from Escherichia coli will be shown.
First, the cell wall is digested by treating with lysozyme for a short time, and RNase to degrade RNAs of Escherichia coli is added. Next, a mixed solution of NaOH and sodium dodecylsulfate (SDS) is added for the purpose of dissolving the cytoplasmic membrane. NaOH partially denatures DNAs and partially degrades RNAs and SDS acts to dissolve the membrane and denature proteins. Successively, SDS-protein complex and cell debris are precipitated by adding 5N potassium acetate (pH 4.8). At this time, pH is important for both to neutralize NaOH used in said manipulation and to renature plasmid. Thereafter, centrifugation is applied to remove the precipitates, thus obtaining aiming plasmids in supernatant.
In a series of these manipulations (hereinafter referred to as pretreatment process), it is important to mix slowly and firmly. If adding violent vibration during this manipulation, then the bacterial chromosomal DNA is cut off to small fragments so that they cannot aggregate, causing them to contaminate the plasmid.
Successively, isopropanol is added to the supernatant, and the mixture is centrifuged to precipitate and concentrate plasmids. Finally, protein is removed from plasmid fraction by precipitating with phenol and chloroform, and plasmid is precipitated with alcohol.
Through a series of manipulations as described above, it is possible to obtain plasmid with relatively high purity (hereinafter, said method of separating and purifying nucleic acid is referred to as chemical separating method). However, with the chemical separating method, separating and purifying process is complicated and a large quantity of organic solvent must be used, hence it poses many problems of treatment of waste solvents and others.
Besides the chemical separating and purifying method, there is a method of separating plasmids by electrophoresis. This method is a technique having the highest resolution at the moment. The electrophoretic method includes paper electrophoresis and gel electrophoresis, and gel electrophoresis is common currently. The electrophoretic method has an advantage of obtaining plasmid with very high purity, while it has many problems of long separation time, difficult collection, low sample loading, etc. Consequently, it is a present situation that the electrophoretic separation is used only when the purity of plasmid fraction purified by said chemical separating and purifying method is desired to improve further.
For solving the problems in chemical separating and purifying method and electrophoretic separation as explained above, a method of separating and purifying nucleic acids that utilizes liquid chromatography has been used recently. So far, there are examples, wherein long chain nucleic acids such as plasmids were separated and purified by using ion exchange chromatography and reversed phase chromatography.
With the method of separating and purifying nucleic acids utilizing liquid chromatography, there are good points of simple manipulation compared with chemical separating method, easy collection of nucleic acids and no necessity of using organic solvent etc. With said conventional method using ion exchange chromatographic method or reversed phase chromatographic method alone, however, there is a problem that the nucleic acids with sufficiently high purity, in particular, long chain nucleic acids such as plasmids cannot be obtained in large quantity.
Therefore, the invention aims at providing a separating method that utilizes liquid chromatography, which enables to separate a large quantity of long chain nucleic acids such as plasmids and DNAs in a shorter time.
The invention of claim 1 of the present application having been made in view of the purpose aforementioned provides a method of separating nucleic acids characterized by using hydrophobic interaction chromatography. And, the invention of claim 7 of the present application provides a method for separating and purifying nucleic acids characterized by using hydrophobic interaction chromatography and ion exchange chromatography in combination.